Project Summary/Abstract: Liquid biopsy (LB) is the analysis of cell-free circulating tumor DNA (ctDNA) in readily-accessible body fluids to non-invasively profile the molecular landscape of solid tumors. Liquid biopsy based on ctDNA can be used to detect actionable mutations, monitor response to treatments and assess the emergence of drug resistance. Liquid biopsy is particularly attractive in non-small cell lung cancer (NSCLC) as activating mutations in Epidermal Growth Factor Receptor (EGFR) confer sensitivity to Tyrosine Kinase Inhibitors(1). However, the analytical sensitivity for liquid biopsy technologies for detecting ctDNA and associated genomic changes is limited by its low concentration compared to cell-free DNA (cfDNA) of non-tumor origin. In 2016, FDA approved the Cobas EGFR Mutation Test v2 using plasma as the first liquid biopsy test for diagnostic use. However, the reported sensitivity for the detection of the 2 most common activating mutations (exon 19 deletions or exon 21 substitutions) in the EGFR gene is only 76.7%(2). Improving the sensitivity of mutation detection could further unlock the potential of liquid biopsies for the diagnosis of cancer including earlier stage detection as well as detection of minimal residual disease. Liquid biopsy analytical platforms that deliver detection sensitivity closest to tissue biopsy-based genotyping of tumor-specific ctDNA is an unmet clinical need. Our preliminary study showed the existence of a novel group of ultrashort circulating tumor DNA (uctDNA) molecules with EGFR mutations in plasma samples from non-small cell lung cancer (NSCLC) patients. This NCI Clinical and Translational Exploratory/Developmental Studies R21 application is to explore and test our hypothesis that there are abundant uctDNA molecules in blood and saliva samples from NSCLC and these uctDNA fragments are additional circulating tumor targets that will improve the sensitivity of liquid biopsy. Two specific aims are in place for hypothesis testing. Aim 1 is to recruit, enroll 250 NSCLC patients that from UCLA Medical Center (UCLAMC) Pulmonary Disease Clinic and VA Greater Los Angeles (VA GLA) Pulmonary Disease Clinic. Plasma will be collected from each patient. Aim 2 is to validate clinical utility of uctDNA NGS assay targeting uctDNA for liquid biopsy of NSCLC. Together, the translational and clinical validations, targeting uctDNA for liquid biopsy can break new ground and extend previous discoveries towards impactful new directions and clinical applications.